The enhanced ability of peripheral mononuclear cells differentiating into neural cells in term infants with good improvement suffering from severe hypoxic ischemic encephalopathy.

OBJECTIVE
It has been found that asphyxia influences proliferation and differentiation of brain neural stem cells in newborn animal models, and that peripheral blood stem cells play an important role in repairing brain damage. But it has not been reported yet whether asphyxia influences peripheral blood stem cells differentiating into neural cells, and whether with the progress of the disease there is a change of peripheral blood stem cells differentiating into neural cells in newborns with hypoxic ischemic encephalopathy (HIE).


METHODS
Fifty term HIE infants were enrolled in research from March, 2007 to March, 2010. There were 10 cases of the severe HIE patients with good improvement, the severe HIE patients with poor improvement, the moderate HIE patients, the mild HIE patients and the controls, respectively. The peripheral mononuclear cells collected within 24 hours and on 7th day after birth were cultured in vitro for 10 days to differentiate into neural cells. The induced nestin positive cells were identified with Immunohistochemistry and counted. Findings : Within 24 hours after birth, there were no difference of induced nestin positive cells among the severe HIE patients with good improvement (68.99±7.85), the severe HIE patients with poor improvement (71.43±6.88), the moderate HIE patients (73.34±6.46), the mild HIE patients (70.46±6.66) and the controls (71.13±7.19, F=0.51, P=0.7). In the severe HIE patients with obvious improvement, the induced nestin positive cells from 7th day peripheral blood mononuclear cells (94.50±15.57) increased markedly compared with that within 24 hours (68.99±7.85, t=4.66, P<0.001), and were higher than the induced nestin positive cells from 7(th) day peripheral blood mononuclear cells in the severe HIE patients with no obvious improvement (94.50±15.57 vs 69.48±5.32, t=4.62, P<0.001).


CONCLUSION
The ability of peripheral mononuclear cells differentiating into neural cells in term infants with good improvement suffering from severe HIE was enhanced, which may suggest possible relationship between the brain repair and the peripheral stem cells.


Introduction
Neonatal hypoxic ischemic encephalopathy (HIE) is one of the most common causes of death and long-term neurological impairment in full-term neonates worldwide. Up to now, there is no specific effective treatment for the severe HIE. The potential use of stem cells to reduce brain damage is a possibility [1] . It has been found that asphyxia influences proliferation and differentiation of brain neural stem cells in newborn animal models [2][3][4] , and that peripheral blood stem cells play an important role in repairing brain damage [5] . But It has not been reported yet whether asphyxia influences peripheral blood stem cells differentiating into neural cells, and whether with the progress of the disease there is a change of peripheral blood stem cells differentiating into neural cells in newborns with HIE. This research will focus on these questions.

Subjects and Methods
Fifty term newborn infants from the pediatric department of Shanghai Tongji hospital affiliated to Tongji University, Shanghai, China, were enrolled into the study from March 2007 to March 2010. 72 hours after birth, the severity of HIE was evaluated according to The Sarnat Grading Scale of HIE, and 20 serious cases, 10 moderate cases, and 10 mild cases were identified. All the patients met the HIE diagnostic criteria. 10 severe cases improved obviously 14 days after birth, which were defined as no consciousness, no seizure, no abnormal muscle tone, no brain stem symptom, whereas 10 severe cases poorly improved, which were defined as having one or more of the above manifestations 14 days after birth or died. 2 cases died within 1 week, and 6 cases died after 1 week. 10 newborn infants with wet lung acted as controls, presenting with breath difficulty, but oxygen saturation was normal. The study protocol was reviewed and approved by the local institutional ethics committee and written informed consent was obtained from all guardians prior to the study Blood sample collection: 5 ml peripheral blood was collected from all newborn infants within 24 hours after birth. Again 5 ml blood was collected from severe HIE patients on 7 th day after birth if the patients were still alive. The mononuclear cells were separated and collected with Ficoll separating medium (GIBCO ) 1500r/min×5min, re-suspended in proliferation culture medium (see below), which were applied in the following process.
The differentiation culture of peripheral mononuclear cells (PMC) [6] : After proliferation culture of peripheral mononuclear cells for 3 days, the culture medium was eliminated, and sterilized PBS was added, then PMCs were gently repeatedly beaten by pipette so that the unattached cells could be eliminated. 2 wells were again continually added with proliferation culture medium as negative controls, and the other 4 wells were added with changed culture medium, namely differentiation culture medium, which consisted of DMEM/F12 (GIBCO), 10% fetal bovine serum (GIBCO), penicillin (Shanghai new pioneer pharmaceuticals company) 100U/ml, streptomycin (Northern China Pharmaceuticals Co) 100µg/ml, all trans retinoid acid (Sigma Co) 0.5 µM, nerve growth factor (Sigma Co) 100ng/ml, insulin (Bo-Yun Biotech) 25 µg/ml, transferring (Bo-Yun Biotech) 100µg/ml, heparin (Wanbang Pharmaceuticals Co) 2 µg/ml, progesterone (Shanghai Pharmaceutical Co Ltd) 60 nM. PMCs were cultured in a humidified atmosphere (37°C, 5% CO2), and half volume of culture medium in each well was renewed with the same medium with the original medium in the well (Proliferation culture medium or differentiation culture medium) every 3 days. The cell shape and growth were daily observed under inverted phase contrast microscope. On the 10th day of differentiation culture, the immunohistochemistry was carried out to identify the differentiated cells.

Immunohistochemistry identification:
Cells in each well were fixed with 4% paraform for 25 min, washed thoroughly with PBS 5 times, permeabilized with 1g/L Triton for 15 min, again washed thoroughly with PBS 5 times, rinsed with

Discussion
HIE is one of the main causes of death and longterm neurological impairment in full-term neonates. There is no specific effective treatment for the severe HIE. Many investigations have identified endogenous neural stem cells were mobilized when HIE took place, namely theneural stem cells in brain take part in HIE repair [1] , and there are also investigations revealing that peripheral blood stem cells have close relation to the damage repair of HIE brain [5,[7][8][9][10][11] Umbilical mononuclear cells consist of multipotent stem cells [12] and mesenchymal stem cells [13] with neuron differentiation potential. Umbilical blood is a part of the neonatal peripheral blood, so it is likely that the neonatal peripheral blood cells possess similar character of week, and the data on 7th day was absent, so the 2 cases were deleted when the data within 24 hrs after birth was analyzed only among the alive patients on 7 th day the umbilical blood cells, namely consist of stem cells with neuron differentiation potential. Our data confirmed that PMCs from newborns and newborns with HIE could differentiate into cells with neural marker namely possessing neuron cells differentiation potential. Human peripheral CD14+ monocytes [14] and CD133+ stem cells in umbilical blood [15] can differentiate into cells with neural markers. It needs to be further confirmed which PMCs gradients in HIE newborn patients can differentiate into neuron cells.
The influence of asphyxia on neonatal peripheral blood stem cells differentiating into neuron cells has not yet been reported by now. Ischemia and anoxia have definite influence on neural stem cells in brain [2][3][4]16,17] . We could not find the influence of asphyxia on the ability of neonatal PMCs differentiating into neural cells, which may suggest the different influence of asphyxia on central neural stem cells and peripheral blood stem cells with neuron differentiation potential.
We found the ability of PMCs from severe HIE with good improvement on 7th day after birth to neural cells obviously increase, which may suggest these patients predominate at promoting neural cells differentiation, and may also suggest peripheral blood stem cells play a possible role in the HIE repair. The mechanism of peripheral blood stem cells taking part in central neural cells repair is unclear, which may relate to directly entering central nervous system to differentiate into neural cells to replace the injured brain tissues or to produce neurotrophin to promote the proliferation and differentiaton of brain internal neural stem cells [18][19][20][21][22][23][24][25][26][27] . The different differentiation ability of PMCs with the progress of HIE has close relationship with the prognosis. If the differentiation ability of PMCs can be identified as early as possible by improving the culture methods to shorten the culture time, it may be valuable to judge the prognosis.
The ability of peripheral mononuclear cells differentiating into neural cells in term infants with good improvement suffering from severe HIE was enhanced, which may suggest possible relationship between the brain repair and the peripheral stem cells. The mechanism of PMCs taking part in the protection of central nervous system injury suffered from asphyxia need to be further investigated.

Conclusion
The ability of peripheral mononuclear cells differentiating into neural cells in term infants with good improvement suffering from severe hypoxic ischemic encephalopathy was enhanced, which may suggest possible relationship between the brain repair and the peripheral stem cells.